Preparation of a Novel Streptokinase Mutant with Improved Stability

Summary

The novel mutant streptokinase, SK-K59E, can activate human plasminogen as efficiently as the purified commercially available streptokinase. Several peptide bonds including Lys⁵⁹-Ser⁶⁰ in native streptokinase were hydrolyzed in reaction with plasmin and peptides of small molecular masses were generated. The plasminogen activator activity of native streptokinase in reaction with human plasmin declined to 25% of the original activity in a 120-min incubation. On the other hand, the NH₂-terminal peptide of SK-K59E remained intact in reaction with plasmin and the activator activity of streptokinase decreased to 75% of the original activity in 120 min. The major degraded peptide fragments of native streptokinase in reaction with plasmin had molecular masses of 36 and 30 kDa. However, two major peptide fragments of 42 and 34 kDa were observed in the reaction of SK-K59E with human plasmin. The 42 kDa peptide fragment, which contained NH₂-terminal of streptokinase, could activate human plasminogen as efficiently as the native streptokinase. SK-K59E can induce greater degree of caseinolysis and fibrinolysis than the native streptokinase. In conclusion, the results demonstrate that the prevention of cleavage at Lys⁵⁹ of streptokinase prolongs the half-life of streptokinase in complex with plasmin and that the NH₂-terminal of streptokinase (Ile¹-Lys⁵⁹) plays an important role in maintaining its stability.

³ Present address: Dung-Ho Wu, Department of Pharmacy, Chia Nan College of Pharmacy and Science, Tainan, Taiwan, 710, R. O. C.

• References

• 1 Jackson KW, Tang J. Complete amino acid sequence of streptokinase and its homology with serine proteases. Biochemistry 1982; 21: 6620-5.
• 2 Reddy KNN. Streptokinase – biochemistry and clinical application. Enzyme 1988; 40: 79-89.
• 3 Shi GY, Chang BI, Chen SM, Wu DH, Wu HL. Function of streptokinase fragments in plasminogen activation. Biochem J 1994; 304: 235-41.
• 4 Young KC, Shi GY, Chang YF, Chang BI, Chang LC, Lai MD, Chuang WJ, Wu HL. Interaction of streptokinase and plasminogen: studied with truncated streptokinase peptides. J Biol Chem 1995; 270: 29601-6.
• 5 Siefring Jr GE, Castellino FJ. Interaction of streptokinase with plasminogen. Isolation and characterization of a streptokinase degradation product. J Biol Chem 1976; 251: 3913-20.
• 6 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-6.
• 7 Brockway WJ, Castellino FJ. Measurement of the binding of antifibrinolytic amino acids to various plasminogen. Arch Biochem Biophys 1972; 151: 194-9.
• 8 Wu HL, Shi GY, Bender ML. Preparation and purification of microplasmin. Proc Natl Acad Sci USA 1987; 84: 8292-5.
• 9 Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988; 16: 7351-69.
• 10 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74: 5463-7.
• 11 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
• 12 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4.
• 13 Wohl RC, Summaria L, Robbins KC. Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37° C. J Biol Chem 1980; 255: 2005-13.
• 14 Shi GY, Chang BI, Wu DH, Ha YM, Wu HL. Activation of human and bovine plasminogen by the microplasmin and streptokinase complex. Thromb Res 1990; 58: 317-29.
• 15 Wu HL, Chang BI, Wu DH, Chang LC, Gong CC, Lou KL, Shi GY. Interaction of plasminogen and fibrin in plasminogen activation. J Biol Chem 1990; 265: 19658-64.
• 16 Wu HL, Wu IS, Fang RY, Hau JS, Wu DH, Chang BI, Lin TM, Shi GY. The binding of plasminogen fragments to cultured human umbilical vein endothelial cells. Biochem Biophys Res Commun 1992; 188: 703-11.
• 17 Radek JT, Castellino FJ. Conformational properties of streptokinase. J Biol Chem 1989; 264: 9915-22.
• 18 Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. Science 1983; 222: 1129-32.
• 19 Malke H, Roe B, Ferretti JJ. Nucleotide sequence of the streptokinase gene from Streptococcus equisimilis H46A. Gene 1985; 34: 357-62.
• 20 Misselwitz R, Kraft R, Kostak S, Fabian H, Welfle K, Pfeil W, Welfle H. Limited proteolysis of streptokinase and properties of some fragments. Int J Biol Macromol 1992; 14: 107-16.